JP2019526531A - Stimulation of platelet production by activation of mitochondrial organisms - Google Patents

Abstract

A method of stimulating platelet formation using a drug that stimulates mitochondrial biogenesis is provided. The drug can be used to treat subjects with thrombocytopenia or a relatively low platelet count, or can be used to stimulate platelet production in vitro or ex vivo. Low level light (LLL) therapy can be used with drugs to stimulate mitochondrial biogenesis. [Selection] Figure 8

Description

Detailed Description of the Invention

(Related application)
This application claims the rights of US Provisional Patent Application No. 62 / 355,027, filed Jun. 27, 2016, the entire contents of which are hereby incorporated by reference.

  Thrombocytopenia causes uncontrollable bleeding and death due to the unusually low platelets in the blood. Until now, the disease has been mainly managed by infusion of platelets, but platelet infusion is associated with multiple types of complications and is limited to patients with life-threatening diseases. In the past 20 years, considerable progress has been made in the development of therapeutics to treat thrombocytopenia, and almost all of these drugs produce megakaryocytes independent of circulating platelet counts. Enhance the growth and differentiation of hematopoietic stem cells (HSC) and / or progenitor cells. Hallam et al., Expert. Opin. Biol. Ther. 13, 1173-1185 (2013). Therefore, high doses of these drugs cause harmful thrombus formation, while low doses show moderate or weak effects, which severely limit their widespread clinical application. An effective method with a low risk of thrombus formation is still an urgent and unsatisfactory medical need for coping with thrombocytopenia.

  Platelets are small anucleated cells that are generated from megakaryocytes (MK), which are mainly present in adult bone marrow (BM), neonatal liver and BM. The cell differentiates from HSC and represents the largest cell (50-100 μm), although it is also one of the rarest cells consisting only of about 0.05% nucleated BM cells under physiological conditions. In patients with thrombocytopenia, the number of cells grows exponentially. During MK maturation, if the cell is not dividing, multiple DNA replications occur, this process is called nuclear division, in which the cytoplasm is extensively amplified and genomic DNA is Amplifies up to 64N, or up to 256N in mice, and at the same time synthesizes abundant cytoskeletal proteins, platelet-specific granules and invaginated membrane systems (IMS). Following cell amplification, proplatelet formation occurs, where terminal mature MK converts all its cytoplasm into many long, branched proplatelets, which are 250-500 μm long within a few hours. Stretch at a rate of about 1 μm / min until it reaches. Patel et al., J. Clin. Invest 115, 3348-3354 (2005). Massive remodeling of cytoplasm and vigorous protrusion and elongation of proplatelets are driven by microtubule forces and are highly dependent on ATP production. This means that mitochondria play a pivotal role in this process. Consistent with this, ultrastructural abnormalities and dysfunction of MK mitochondria are usually associated with the production of impaired platelets in patients with myelodysplastic syndrome (MDS) and immune thrombocytopenia (ITP). Point mutations in mitochondrial cell cytochrome c cause dysregulated platelet formation and thrombocytopenia, especially in humans, indicating that platelet biosynthesis is very sensitive to mitochondrial activity. In recent studies, mitochondrial dysfunction tends to lack the immediate early response gene X-1 (IEX-1) for thrombocytopenia when mice are exposed to stress. Ramsey et al., Haematologica 99, 282-291 (2014). One of the main functions of IEX-1 is to enhance ATP synthase activity in the mitochondrial respiratory chain, and its invalid mutation compromises the production of ATP and in a cell type specific manner Increases production of reactive oxygen species (ROS) in mitochondria. The ability of the mitochondrial specific antioxidant mitoquinone to completely reverse thrombocytopenia in IEX-1-deficient mice clearly indicates that mitochondrial function is crucial in platelet production. Ramsey et al., Platelets, 26 (5): 459-66 (2015).

  In one aspect, the present application provides a method of stimulating platelet formation in a subject's body by administering to the subject an effective amount of a drug that stimulates mitochondrial biogenesis. In the course of studying the mechanism of low-level light (LLL) -mediated platelet biogenesis, the inventors have found that LLL enhances mitochondrial biogenesis, primarily in megakaryocytes, resulting in increased platelet formation. I found Mitochondrial mass and ATP production in megakaryocytes are proportionally correlated with the level of platelet formation.

  A number of drugs that promote mitochondrial biogenesis are known. Examples include p38 mitogen activated protein kinase activator, calmodulin-dependent protein kinase IV activator, AMP activated kinase activator, calcineurin A activator, peroxisome proliferator response element activator and SIRT1 activation Agents.

  In some examples, the subject treated with a drug that stimulates mitochondrial biogenesis has been diagnosed as having thrombocytopenia. In some other examples, the method includes administering an antithrombocytopenic agent to the subject. In another embodiment, the method includes treating the subject with low level light (LLL) therapy. In a further embodiment, the drug is administered with a pharmaceutically acceptable carrier.

  In another aspect, the invention provides a method of stimulating platelet production, comprising contacting a platelet precursor with a drug that stimulates mitochondrial biogenesis. In some embodiments, the platelet precursor is a megakaryocyte or megakaryoblast. In some other examples, the platelet precursor is in vitro or ex vivo. In other examples, platelet precursors are also exposed to low level light therapy.

A to H are graphs and images showing that LLL promotes MK maturation and platelet production. (A) is a schematic diagram of a timeline of ex vivo platelet differentiation from MK. CD41 + FSC ^high MK is selected from BM cells, treated with or without LLL, and cultured in MK medium. After 1 hour, collect MK for measurement of cellular ATP (B), 1 day later, collect MK to study proplatelet formation (CF), or 3 days later to count platelets Collect MK (G). (B) Measure ATP at 5 × 10 ⁴ MK for LLL treatment with different energy densities. (C) Analyze CD41 + MK size by forward / side scatter (FSC) flow cytometry analysis before and after 24-hour differentiation. (D) is a representative transmission electron micrograph of MK of at least 6 samples per group (with 30 cells in each group). N is the nucleus and IMS is the invaginated membrane system. (E) is a representative image of PPF-MK 24 hours after LLL. The PPF-MK diameter satisfies <100 μm when small and ≧ 100 μm when large. The black triangles indicate one of the many protrusions on the proplatelet shaft (proplatelet shaft). Unfilled triangles indicate nuclei. (F) The numbers indicate the percentage of small or large PPF-MK in at least 500 MKs for each sample analysis, with 6 samples per group. ### P <0.001, large PPF-MK, and ** P <0.01, total PPF-MK compared between the two groups. (G) Based on CD41 expression and FSC, the number of platelets derived from 1 × 10 ⁴ MK is estimated on the third day after LLL. (H) Sorted MKs are treated with or without LLL, labeled with CFSE, and injected into recipient mice at 1 × 10 ⁵ cells per mouse. Shown is the percentage of CFSE + platelets obtained in the recipients for the specified number of days. All data are expressed as mean ± SEM, where n = 6 for B, C and G, or n = 10 for H. Compared to the control, *** P <0.001. The scale bar is 5 μm at D or 25 μm at E.

A to H provide graphs and images showing that the platelet producing action of LLL is ATP-dependent. (A) The correlation between the MK ATP level 1 hour after LLL and the platelets measured 3 days later is analyzed with a coefficient of determination. (B and C) 5 μg / ml inhibitor oligomycin A (OA) suppresses LLL from affecting platelet production (B) and ATP synthesis (C). (D) WT and IEX-1 KO BM cells are stained with anti-CD41 antibody and JC1. Determine the mitochondrial membrane potential of CD41 + FSC ^high MK by flow cytometric analysis of red J-aggregate fluorescence at 590 nm. (E) The selected MK is processed with or without LLL, and differentiated for 1 hour before ATP measurement as shown in FIG. 1B. (F and G) Representative images of PPF-MK are obtained from at least 6 samples per group (with 25 cells per group) 24 hours after LLL (F). The scale bar is 25 μm. The cell diameter is indicated by G, where each symbol represents a single PPF-MK. (H) Based on CD41 expression and FSC (H), the number of platelets derived from 1 × 10 ⁴ MK is estimated 3 days after LLL. All data represent mean ± SEM. n = 6, compared between the specified groups, *** P <0.01 and *** P <0.001.

A-L provide graphs and images showing that LLL stimulates mitochondrial biogenesis in polyploid MK. (A) After LLL, ATP in MK, BM cell or LSK is measured at a specified number of times. (B and F) 24 hours after LLL, the cells designated with MitoTracker are stained and analyzed by flow cytometry. (C) Measure mitochondrial DNA content of MK by real-time PCR and normalize with nuclear DNA. (D, G and H) PGC-1α transcripts are measured 4 hours after LLL (D sum G), and other gene transcripts are measured by qRT-PCR 16 hours after LLL (D sum H). (E to I) MK was selected based on DNA content by staining with Hoechst 33342 and FITC-anti-CD41 (E), treated with LLL or sham light, and 24 hours later using MitoTracker Perform flow cytometry analysis (F), or perform RT-qPCR analysis of PGC-1α transcripts 4 hours after LLL (G), and transfer other genes 16 hours after LLL as described above. The product (H) is analyzed. The number of platelets derived from 1 × 10 ⁴ MKs is estimated 3 days after LLL (I). (J to L) A typical transmission electron micrograph of MK 24 hours after LLL is shown in (J). The scale bar is 5 μm. Image J software measures the mitochondrial number (K) of each MK and the shortest distance (L) between each mitochondria and the nearest nuclear region from at least 30 MKs per group. All other data are from 3 independent experiments, each experiment repeated 3 times, expressed as mean ± SEM and compared to non-LLL controls, * P <0.05 and ** * P <0.001.

A-D provide graphs and images showing that LLL penetrates into the bones of mice. (A) Measure the transmittance (%) of the specified LLL mode under the fresh skin and vertebrae of the mouse using a laser power meter. (B) To measure ATP levels as in FIG. 1B, BM cells are separated from designated bones 1 hour after 30 J / cm ² whole body LLL irradiation. (C and D) Mice are injected intravenously with FITC-anti-CD41 (green) and PE-anti-CD105 (red) antibodies 24 hours after whole body LLL irradiation and die 15 minutes later. Subsequently, a fresh femur is removed from the mouse and examined with a confocal microscope (C). The arrow indicates BM MK. The scale bar is 50 μm. The percentage of PPF-MK is determined from at least 50 MKs per femur and 6 samples per group (D). * P <0.05, ** P <0.01 and *** P <0.001 compared to control or between designated groups, and n = 6 (A to C ).

AH provide graphs and images showing that LLL attenuates IR-induced thrombocytopenia in vivo. (A) Total blood counts of white blood cells (WBC), lymphocytes, monocytes, granulocytes and red blood cells (RBC) in mice that were irradiated with 3-Gy γ-irradiation 2 weeks after IR. Data are mean ± SEM (n = 15). (B) Obtain platelet counts in mice (IR) irradiated with 3-Gyγ-irradiation on designated days, or platelet counts in IR mice treated once with LLL 6 hours after IR (IR + 1 × LLL Or obtained once a day (IR + 2 × LLL) on days 0 and 1 or once a day (IR + 4 × LLL) from day 0 to day 3. Data are mean ± SEM (n = 15). For IR, * P <0.05, ** P <0.01, *** P <0.001. (C) Two weeks after IR, the bleeding time of the tail of each mouse was examined and represented by individual symbols. (D) The platelet volume of each mouse is examined 2 weeks after IR. All the data are mean ± SEM (n = 10). (E) Representative transmission electron micrograph of platelets isolated from a designated mouse 2 weeks after IR. The scale bar is 1 mm. (F) In platelets isolated from non-IR control and IR + 4 × LLL mice 2 weeks after LLL, labeled with anti-CD9 or anti-CD31 antibody, mixed, phorbol 12-myristate 13-myristate (phorbol 12- Stimulate with myristate 13-acetate (100 ng / ml). Flow cytometry quantifies the aggregated platelets indicated by double color events and shows the mean percentage ± SEM (n = 6). (G) Treat circulating platelets and BM MK in mice for 12 days at 30 J / cm ² with LLL every other day to maintain within stable levels. Data are mean percentage change relative to baseline ± SEM (n = 6). (H) Effects of 4 doses of LLL over time on the number of MKs in γ-irradiated mice. Data are mean MK ± SEM (n = 6) for each femur in the specified number of days. Compared to the IR group, * P <0.05 and *** P <0.001. P values are determined by two-tailed Student's t test (A and H) or one way ANOVA (B to D).

AD provides graphs showing that LLL reduces thrombocytopenia induced by anti-CD41 antibody or 5-FU in mice. (A and B) Mice receive 0.1 mg / kg anti-CD41 antibody daily for 7 days. From day 3 to day 7, LLL is given daily. (C and D) On day 0, mice are injected with 50 mg / kg of 5-FU. LLL is given daily for 4 consecutive days after 5-FU injection for 3 consecutive days. Platelet counts are measured daily 6 hours after LLL (A and C) and tail bleeding time is examined on days 5 (B) or 4 (D) (B and D). n = 6, ns, not significant. Comparing between the specified groups, * P <0.05, ** P <0.01, *** P <0.001.

AD provides images and graphs showing that LLL significantly enhances platelet production in human MK. (A) Timeline of ex vivo platelet differentiation from human CD34 + cells. CD34 + cells differentiate mainly into MK progenitor cells, mature MKs, and platelets on day 6, 12, or 15 respectively. (B) As shown in FIG. 1B, ATP is measured in MK derived from CD34 + treated with LLL of different energy density. (C) Ploidy analysis is performed on CD34 + cultures on day 0, 6 or 12 using Hoechst 33342 staining. (D) Administer 3 J / cm ² LLL on day 0, 6 or 12 during CD34 + cell differentiation. Platelets are quantified by flow cytometry on day 15 and are expressed as mean values ± SEM of platelets derived from 1 × 10 ⁴ CD34 + cells. All data are obtained from three independent experiments, and each experiment is repeated three times. * P <0.05 and ** P <0.01 compared to the control.

A strategy for drug-induced PGC-1α-mediated mitochondrial biogenesis is provided. Nuclear respiratory factors (NRF-1 and NRF-2) control all ten nuclear-encoded cytochrome oxidase subunits. For ERR, the estrogen-related receptors α, β, γ, for TCA, the tricarboxylic acid cycle, and for FAO, fatty acid oxidation.

1 provides a graph showing that mitochondrial drugs (mito-drug) enhance ex vivo platelet production. For C, it is a control, for Bez it is bezafibrate (400 μM), for Res it is resveratrol (50 μM), for SRT1720 it is 0.1 μM, for AicAR it is AICAR (500 μM), and for LLL it is 810 nm 3 J / cm ² . Mouse MKs are allowed to differentiate for 3 days in the presence of the specified drug in MK medium or after LLLT. ***, p <0.01 and ***, p <0.001 compared to the control. n = 6.

1 provides a graph showing enhanced plt production by mitochondrial drugs in vivo. All mice are treated with two doses of 5-FU (FU) for 4 consecutive days with the designated mitochondrial drug or LLLT (red arrow). *, P <0.05, **, p <0.01 and ***, p <0.001 in the presence or absence of the specified drug or LLLT. In each group, n = 10.

FIG. 7 provides a diagram showing the enhancement of platelet production by mitochondrial drug + LLLT in vivo. All mice are treated daily with 0.1 mg / kg with anti-CD41 antibody for 7 days. Mice were treated with LLLT (980 nm, 0.025 J / cm ² once daily for 4 consecutive days (red arrow)) and BEZ (100 mg / kg twice daily for 4 consecutive days). Process) or both. ***, p <0.001 compared to the control. In each group, n = 10.

  The present invention provides methods of stimulating platelet formation using drugs that stimulate mitochondrial biogenesis. The drug can be used to treat subjects with thrombocytopenia or diagnosed with a relatively low platelet count or used to stimulate platelet production in vitro or ex vivo. Can do. Low level light (LLL) therapy can be used with drugs to stimulate mitochondrial biogenesis.

  The terminology described herein is for the purpose of describing examples only and should not be construed as limiting the invention as a whole. Unless otherwise specified, “one”, “above”, “the relevant” and “at least one” can be used interchangeably. Further, as used in the description of the invention and the appended claims, the singular forms “a”, “the”, and “the” are used to refer to the plural numbers unless the context dictates them. Includes shape.

  In addition, in this specification, the numerical value range described at the end point includes all the numerical values included in the range (for example, 1 to 5 is 1, 1.5, 2, 2.75, 3, 3 .80, 4, 5, etc.).

  As used herein, the term “subject” includes any human, rat, mouse, dog, goat, sheep, horse, monkey, ape, pig, rabbit, cow, etc. Can refer to warm-blooded organisms. Where a subject requires or requires the use of this term in the context of a composition of the present application, the term is referred to as “subject in need thereof” and the subject in need of the composition of the present application, the present application A subject suspected of needing a composition, a subject who is at risk of having a disease or illness, and a subject and a disease or illness who may be endowed with the composition of the present application, and the composition of the present application Includes subjects who may be blessed with things and clinically diagnosed (eg, by a medical professional, eg, a physician).

  As used herein, the term “pharmaceutically acceptable” refers to excessive toxicity, irritation, allergic reactions, or other problems or complications within the scope of reliable medical judgment. Refers to those compounds, materials, compositions and / or dosage forms suitable for use in contact with human and animal tissue at conditions commensurate with a reasonable benefit / risk ratio.

  As used herein, the term “diagnosis” can include determining the likelihood that a subject will develop a disease, or the presence or nature of a disease in a subject. As used herein, the term “diagnosis” further includes determining the severity of the disease or disease onset and the expected outcome or likelihood of recovery (commonly referred to as prognosis).

  As used herein, the term “treatment” or the like refers to obtaining a desired pharmacological or physiological effect. With respect to partial or complete cure of a disease or side effects resulting from the disease, the effect can be therapeutic. As used herein, “treatment” includes any treatment of a disease in a mammal (particularly a human) and suppresses the disease or condition, ie, inhibits its progression, and the disease May be included, i.e., alleviating the disease.

  As used herein, the term “prevention” refers to completely preventing the development of a clinically apparent disease (eg, bleeding), or an apparent preclinical stage of disease in a subject (eg, Prevention of the development of thrombocytopenia). Prophylactic treatment is particularly useful in subjects who have been determined to be at high risk of developing the disease. A high risk indicates that the subject has a higher than average level of risk for developing a disease such as thrombocytopenia. Examples of factors that represent a high risk of developing thrombocytopenia include vitamin deficiency, leukemia, sepsis, liver failure, and high platelet destruction rates are known to induce immune or non-immune diseases, myelosuppression May be due to treatment with certain drugs (eg valproic acid or methotrexate), snake bite, radiation, radiation therapy for cancer patients, chemotherapy or chemo / radiotherapy and Lyme disease.

  The term “therapeutically effective” reduces the severity of the disease and reduces the amount of each drug to achieve the goal of avoiding adverse side effects (eg, poor side effects usually associated with alternative therapies). It is intended to be limited. A therapeutically effective amount can be administered in one or more doses. If the term is included within the meaning of the term used herein, a therapeutically effective treatment improves the quality of life of the subject without itself improving the outcome of the disease. Including treatment.

  An “effective amount” is generally an amount that acts to effectively stimulate platelet formation including the desired local or systemic effect, eg, exerting a particular desired effect described herein. Point to. For example, an effective amount is an amount sufficient to achieve a beneficial or desired clinical result.

  As used herein, contact means that two objects are physically adjacent and in contact, or that they are placed in an environment where such contact occurs within a reasonable amount of time. To do. For example, contacting a cell with a drug that stimulates mitochondrial biogenesis administers the drug to a subject such that the drug interacts with the cell at the site (lung, bone marrow, spleen and liver) to stimulate platelet production. Including that. However, contact further includes systemic administration that results in contact between the drug and the platelet precursor via circulation-mediated contact.

All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. The definitions provided herein are for ease of understanding certain terms frequently used herein and are not meant to limit the scope of the present application.
Stimulates platelet biogenesis

  The present invention provides a method of stimulating platelet formation in a subject by administering to the subject an effective amount of a drug that stimulates mitochondrial biogenesis. The normal platelet count range is 150,000 to 450,000 platelets per microliter of blood. In some embodiments, the subject is a subject in need of treatment because it has a small number of platelets. For example, about 5-15% of normal platelet levels, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85% or 85-85 Subjects with 95% may need treatment. Subjects with 40-95% of normal platelet levels are generally not considered to have thrombocytopenia but can benefit from stimulation of platelet formation. By placing the blood in various automated blood analyzers, the platelet concentration (because it is too small to count) is measured using electrical impedance or flow cytometry.

  In some embodiments, the subject has already been diagnosed with thrombocytopenia. Thrombocytopenia is one type of disease and the subject has an abnormally small amount of platelets, for example, less than 50000 platelets per microliter, or is normal (average or median) for a particular human Have platelets lower than 2.5% of the platelet count. Thrombocytopenia generally does not show symptoms, but patients with thrombocytopenia may show symptoms such as nasal or gum bleeding, bruising (purpura) and increased external bleeding such as fatigue. Thrombocytopenia can be inherited or is caused by a number of different diseases known to those skilled in the art (eg, pyoderma or lupus).

The present invention provides a method of stimulating platelet formation. Platelets (also called clotting cells) are one of the components of blood and their function (along with clotting factors) is to stop impaired vascular bleeding by aggregation and coagulation. Platelets have no cell nucleus and are cytoplasmic fragments derived from megakaryocytes. In stained blood smears, platelets appear as dark purple spots, about 20% of the red blood cell diameter. Stimulation of platelet formation refers to improving the rate of platelet formation by megakaryocytes. Stimulation is 1-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90- It can refer to an increase of 100%, 100-150%, 150-200% or greater than 200%.
Mitochondrial biogenesis stimulating drugs

  One aspect of the present invention further comprises administering an effective amount of a drug that stimulates mitochondrial biogenesis. Inventors have already established that mitochondria play an important role in platelet formation by platelet precursors (eg megakaryocytes), so drugs that stimulate mitochondrial biogenesis are used to stimulate platelet formation can do. As used herein, the term “biogenesis” refers to the synthesis of biological material, so the term “mitochondrial biogenesis” refers to the synthesis of mitochondria. Mitochondrial biogenesis can be demonstrated by increased expression of genes associated with mitochondrial biogenesis, which are PGC family members such as PGC-1α and PGC-1β, PPARδ, NRF-1, SIRT1, SIRT3, Including, but not limited to, COX and AMPK. Alternatively, it refers to increased mitochondrial DNA or protein content, higher ratio of mitochondrial DNA to nuclear DNA, or improved mitochondrial function (eg, increased activity of mitochondrial enzymes or increased mitochondrial respiration). Mitochondrial biogenesis stimulating drugs may have a general stimulating effect on mitochondrial biogenesis, but for stimulation of platelet biogenesis, stimulation of mitochondrial biogenesis in platelet precursors (eg, megakaryocytes) is particularly is important.

  In some embodiments, the stimulatory drug for mitochondrial biogenesis is a p38 mitogen-activated protein kinase activator, a calmodulin-dependent protein kinase IV activator, an AMP-activated kinase activator, a calcineurin A activator, a peroxisome Selected from the group consisting of a proliferator response element activator and a SIRT1 activator. Both of these activators have been shown to be able to stimulate increased mitochondrial biogenesis.

  In some embodiments, the drug that stimulates mitochondrial biogenesis is selected from the group consisting of specific compounds known to stimulate mitochondrial biogenesis. Examples of compounds known to stimulate mitochondrial biogenesis include interleukin 15 (US Patent Publication No. 2016/0354442), hydroxymethylbutyrate (US Patent Publication No. 2016/0346238), bioactive alkaloids (US Patent Publication). No. 2016/0184338), rhenium-based carbon monoxide releasing compounds (US Patent Publication No. 2016/0243151), muscadine and resveratrol (US Patent Publication No. 2013/0184228), hydroxytyrosol (US Patent Publication No. 2015/0030579), curcumin compounds (US Patent Publication No. 2015/0297536), flavonoid compounds (US Patent Publication No. 2014/0256741) and β2 adrenergic receptor agonists (US Patent Including the Publication No. 2014/0024677). In other examples, drugs that stimulate mitochondrial biogenesis include bezafibrate, rosiglitazone, pioglitazone, fenofibrate, AICAR, metformin, resveratrol, SRT1720, SRT2183 , As well as their derivatives. As used herein, a derivative refers to a compound having the same basic chemical backbone, such as a particular aromatic heterocyclic compound. However, the part located on the skeleton structure can be changed without losing the activity of the compound. Minor changes include heterogeneous mutations such as the use of different halogens, substitution of oxygen for sulfur, extension of alkyl side chains by a single methyl group, and the like.

Candidate drugs for stimulating mitochondrial biogenesis in animal models can be tested. In general, animal models are animal models for studying thrombocytopenia or mitochondrial biogenesis. For example, see US Patent Publication No. 2005/0177887, which describes a PTTG knockout rodent that can be used as an animal model for thrombocytopenia. The results are generally compared between control animals treated with the candidate agent and control litter pups not receiving treatment. For example, the effects of co-agents on PGC-1α and PGC-1β, PPARδ, NRF-1, SIRT1, SIRT3, COX and AMPK can be tested, or the amount of mitochondrial DNA or protein content, against nuclear DNA A high proportion of mitochondrial DNA, these are all markers of mitochondrial biogenesis. Transgenic animal models are also available and are generally accepted as models of human disease (see, eg, Greenberg et al., Proc. Natl. Acad. Sci. USA, 92: 3439-3443 (1995)). ). Candidate agents can be used in animal models to determine whether candidate reagents stimulate mitochondrial biogenesis.
Combination with antithrombocytopenia

In some embodiments, the method further comprises administering to the subject a second antithrombocytopenic agent in combination with a drug that stimulates mitochondrial biogenesis. Second antithrombocytopenic drugs are drugs that are known to be useful in the treatment of thrombocytopenia. “Anti-thrombocytopenic agents” include recombinant TPO and PEGylated human megakaryocyte growth and development factor (PEG-rhMGDF), and thrombopoietin (TPO), which includes the so-called TPO mimic, and TCP as an agonist of the TPO receptor. It has been used to treat effectively. TPO mimetics include non-peptide molecules and peptides. For example, Nplate ^™ (also called romiprostim, AMG 531) is one of the most well-developed TPO mimics and is a fusion protein of a TPO receptor binding peptide and an IgG1 antibody Fc domain. Eltrobopag is an exemplary non-peptide TPO mimetic.

  US Pat. No. 7,160,870 describes other suitable TPO mimetics that can be used as antithrombocytopenic agents, for example, 3 ′-{N ′-[3-cyclo Propyl-1- (3,4-dimethylphenyl) -5-oxo-1,5-dihydropyrazole-4-ylidene] hydrazino} -2'-hydroxybiphenyl-3-carboxylic acid, [1- (4-fluoro- 3-methylphenyl) -3-methyl-5-oxo-1,5-dihydropyrazole-4-ylidene] -hydrazino} -2'-hydroxybiphenyl-3-carboxylic acid, 3 '-{N'-[3- Methyl-5-oxo-1- (4-trifluoromethylphenyl) -1,5-dihydropyrazole-4-ylidene] hydrazino} -2′-hydroxybiphenyl-3-carboxylic acid, 3- {N ′ − [1 -(3, -Dimethylphenyl) -3-methyl-5-oxo-1,5-dihydropyrazole-4-ylidene-] hydrazino} -2-hydroxy-3'-tetrazol-5-ylbiphenyl, 3 '-{N'-1 -(3,4-Dimethylphenyl) -3-methyl-5-oxo-1,5-dihydropyrazole-4-ylidene-hydrazino} -2'-hydroxybiphenyl-3-carboxylic acid, 3 '-{N'- [1- (3-Fluoro-4-methylphenyl) -3-methyl-5-oxo-1,5-dihydropyrazole-4-ylidene] hydrazino} -2'-hydroxybiphenyl-3-carboxylic acid, 3'- {N ′-[1- (3,4-Dimethylphenyl) -3-ethyl-5-oxo-1,5-dihydropyrazole-4-ylidene] -hydrazino} -2′-hydroxybiphenyl-3- Rubonic acid and 3- {N ′-[1- (3,4-dimethylphenyl) -3-ethyl-5-oxo-1,5-dihydropyrazole-4-ylidene] -hydrazino} -2-hydroxy-3 '-Tetrazol-5-ylbiphenyl is described and 3'- {N'-[1- (3,4-dimethylphenyl) -3-methyl-5-oxo-1,5-dihydropyrazol-4-ylidene] Preferred are hydrazino} -2′-hydroxybiphenyl-3-carboxylic acid and pharmaceutically acceptable salts, hydrates, solvates or esters thereof.

  “Combination” refers to the administration of a drug that stimulates mitochondrial biogenesis and a certain amount of an antithrombocytopenic agent so that there is a cumulative or synergistic effect, and separately, simultaneously or sequentially If no drug is administered and a drug that stimulates mitochondrial biogenesis is administered, the cumulative effect or synergistic effect cannot be obtained. Administration of the anti-thrombocytopenic agent may be continuous, sequential or accidental. Thus, as used herein, the combination should not be limited to a single formulation comprising the combination of the present invention, but a strategy or treatment in which the active agents of the combination of the present invention are administered in different dosage forms. Is open.

The appropriate dose of the drug depends on the route of administration, age, weight, sex, or subject's condition, but is ultimately determined by a physician. For oral administration, the daily dosage is generally about 0.01 mg to about 500 mg per kg body weight, preferably about 0.01 mg to about 50 mg, more preferably about 0.1 mg to about 10 mg. is there. For parenteral administration, the daily dosage is generally from about 0.001 mg to about 100 mg per kg body weight, preferably from about 0.001 mg to about 10 mg, more preferably from about 0.01 mg to about 1 mg. It is. Daily doses can be administered, for example, in a typical manner with 1 to 4 individual doses daily. The dosage can be measured by using the target serum concentration. Various considerations for reaching an effective amount are described, for example, by Goodman and Gilman: The Pharmaceutical Bases of Therapeutics, 8th ed. , Pergamon Press, 1990, and Remington's Pharmaceutical Sciences, 17th ed. Mack Publishing Co. Easton, Pa. 1990.
Pharmaceutically acceptable carrier

  In some embodiments, the drug that stimulates mitochondrial biogenesis is administered with a pharmaceutically acceptable carrier. Other drugs (eg, antithrombocytopenic agents) can also be administered with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include one or more additional ingredients that contribute to administering the drug or improving its pharmacokinetics. Examples of ingredients contained in pharmaceutically acceptable carriers include pharmaceutically acceptable excipients and diluents. Suitable excipients and / or diluents are well known in the art and include pharmaceutical grade starch starch, mannitol, lactose, magnesium stearate, sodium saccharine, talc, cellulose, glucose, sucrose (or other sugar), carbonic acid Contains magnesium, gelatin oil, alcohol, detergent, emulsifier or water (preferably sterile). A pharmaceutical composition comprising a pharmaceutically acceptable carrier is a preservative, solubilizer, stabilizer, wetting agent, emulsifier, sweetener, colorant, odorant, salt, buffer, coating agent or antioxidant. Agents can also be included.

  The drug composition may be administered by any suitable route, such as parenteral, oral (including buccal or sublingual), rectal or topical (including buccal, sublingual, intradermal or transdermal) routes. it can. Such compositions can be prepared by any method known in the pharmaceutical art, for example by mixing the active ingredient with a carrier or excipient under aseptic conditions.

  Pharmaceutical compositions suitable for oral administration are as discrete units such as capsules or tablets, as powders or granules, as solutions, syrups or suspensions (in aqueous or non-aqueous liquids or as edible foams or As a whipping agent or as an emulsion). Suitable excipients for tablets or hard gelatin capsules include lactose, corn starch or derivatives thereof, stearic acid salts thereof. Suitable excipients for soft gelatin capsules include, for example, vegetable oils, waxes, fats, semisolids, or liquid polyols. Excipients that can be used to prepare solutions and syrups include, for example, water, polyols and sugars. To prepare the suspension, an oil (eg, vegetable oil) can be used to provide an oil-in-water or water-in-oil suspension.

  Drug compositions suitable for topical administration can be formulated as ointments, creams, suspensions, detergents, powders, solutions, pastes, gels, sprays, aerosols or oils. Drugs are delivered through the skin by microneedles or microneedle arrays, drug-containing, drug-embedded, or drug-coated microneedle arrays (soluble or non-soluble microneedle arrays, hollow microneedles or microneedle arrays) can do. It can be delivered by insulin pumps, catheters, wearable syringe pumps and micro-delivery technology (this drug acts systemically in the body and requires large quantities so it can be delivered by topical application of the skin Is low). Pharmaceutical compositions suitable for rectal administration can be suppositories or enemas.

Drug compositions suitable for parenteral administration are aqueous and non-aqueous sterile injection solutions that can contain antioxidants, buffers, bacteriostats and solutes that render the formulation substantially isotonic with the blood of the intended receptor And aqueous and non-aqueous sterile suspensions which can contain suspending agents and thickening agents. Excipients that can be used for injection solutions include, for example, water, alcohols, polyols, glycerin and vegetable oils. The composition may be present in unit-dose or multi-dose containers, such as sealed ampoules and vials, and may be stored lyophilized (lyophilized) and applied just prior to use. It is only necessary to add a sterile liquid (eg injection solution). Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets.
Low level light therapy

  In some embodiments, the method includes treating the subject with low level light (LLL) therapy. As used herein, the term “low level light (LLL)” refers to cells (eg, stem cells, other types of platelet progenitor cells, platelets, etc.), tissues and / or at least part of a patient's body. (E.g., adult platelet-forming bones or infant bones and livers) with lower levels of red light and near-infrared (Nr NIR) can refer to a process that is exposed to light. As used herein, the term LLLT (“low level light therapy”) may be used interchangeably with LLL.

Typically, low-level light (LLL) is a cell (eg, at a single dose or multiple doses) at a lower energy density than other forms of laser therapy (eg, ablation, amputation, thermal coagulation, etc.) Stem cells, megakaryocytes, other platelet precursor cells, platelets, etc.), tissues (eg, bone marrow and / or liver), and / or at least part of the patient's body. For example, the LLL energy density may be 0.001 J / cm ² to 30 J / cm ² . As another example, the LLL energy density may be 0.001 J / cm ^{2 to} 20 J / cm ² . As another example, the LLL energy density may be 0.1 J / cm ^{2 to} 0.5 J / cm ² . LLL is a simple, non-invasive, safe, convenient and cost effective method that has been used clinically for decades for pain relief and other applications. In various embodiments, the LLL used herein can have a wavelength of 600 nm to 1500 nm, a wavelength of 600 nm to 1100 nm, or a wavelength of 900 nm to 1000 nm.

Without wishing to be bound by theory, LLL is intended to enhance in vivo and in vitro platelet biogenesis and prolong platelet life because at least LLL can enhance ATP synthesis in cells and / or platelets. It can be used. The inventor has demonstrated that mitochondria are sites where the initial effects of LLL occur. Zhang et al., Sci Transl Med. , 8 (349), 349ra101 (2016) and Example I of this specification. LLL can excite multiple protein complexes (eg, I, III and / or IV) in the mitochondrial respiratory chain (MRC). Normally, MRC can produce more than 90% of ATP in the cell, but LLL increases the amount of ATP in the cell due to a decrease in the level of ATP synthesis in stressed cells. Is possible. In some cases, LLL (additionally or alternatively increases ATP synthesis) can lead to enhanced oxidative phosphorylation, enhanced mitochondrial membrane potential, reduced oxidative stress, and anti-apoptosis.
Methods for stimulating platelet formation

  Another aspect of the invention provides a method of stimulating platelet formation comprising contacting a platelet precursor with a drug that stimulates mitochondrial biogenesis. The method can stimulate platelet formation in vivo, in vitro, and ex vivo. In some embodiments, platelet precursors are also exposed to low level light treatment. The drug that stimulates mitochondrial biogenesis can be any drug described herein.

  “Progenitor cells” are cells that lose most of their pluripotency and become unipotent, partially differentiated cells. As used herein, “unipotent cell” refers to a progenitor cell that differentiates into only one cell type. “Platelet precursor” can refer to any cell that contributes to platelet biosynthesis. These cells are usually found in the bone marrow and / or liver. Examples of platelet precursors include hematopoietic stem cells, pre-megakaryocytes, megakaryocytes, megakaryocytes and the like.

  In some embodiments, the platelet precursor is a megakaryocyte or megakaryocyte. Megakaryocytes are large bone marrow cells with a segmented nucleus responsible for platelet production. Megakaryocytes are 10-15 times larger than typical erythrocytes and have an average diameter of 50-100 μm. During its maturation, megakaryocytes grow in size and replicate their DNA in a process called endomitosis, without cytokinesis. Proplatelet formation occurs following cell expansion, except that terminal mature MK converts all its cytoplasm into many long branched proplatelets, which grow at a rate of about 1 μm / min and within 250 hours It reaches a length of ˜500 μm. Such cytoplasmic remodeling and active protrusion and growth of proplatelets are driven by microtubule forces and are highly dependent on ATP production by mitochondria. Thrombopoietin plays a role in inducing megakaryocytes to form proplatelets.

The following examples are for illustrative purposes only and are not intended to limit the scope of the appended claims.
Example
Example 1: Non-invasive low level laser therapy used for thrombocytopenia

Many researchers, including us, have a special near-infrared laser (referred to as a low-level laser (LLL) or cold laser) with a relatively low energy density that activates cytochrome c oxidase in the mitochondrial respiratory chain and Indicated that it can be improved. Yu et al., Photochem. Photobiol. 66, 866-871 (1997). Zhang et al. Cereb. Blood Flow Metab 34, 1391-1401 (2014). LLL appears to be able to directly improve mitochondrial membrane potential, stimulate ATP synthesis, and regulate cellular ROS and Ca ²⁺ levels. Dong et al. Cereb. Blood Flow Metab. , 35 (9), 1435-44 (2015). LLL can also reduce oxidative stress, prevent cell apoptosis, reduce inflammation, and promote cell proliferation and differentiation. AlGhamdi et al., Lasers Med. Sci. 27, 237-249 (2012). Light irradiation regulates other signaling pathways and more effectively regulates mitochondrial function under various stress conditions. Song et al. Neuroinflamation, 9, 219 (2012). The beneficial effects of LLL on traumatic brain injury have been demonstrated in many preclinical studies. Zhang et al. Cereb. Blood Flow Metab, 34, 1391-1401 (2014). However, the effect of LLL on platelet production is not known at all.

This study shows that non-invasive whole-body LLL irradiation increases platelet production and completely cures thrombocytopenia caused by γ-irradiation, ITP or 5-fluororacyl (5-FU) in mice, or It proved to be a big improvement. Although LLL instantaneously increases ATP production in MK, HSC and BM cells, LLL primarily promotes mitochondrial biogenesis with polyploid MK, specifically targeting MK, but diploid Does not promote mitochondrial biogenesis in cells. This discovery has great expectations that LLL will become a prevention and treatment modality for treating thrombocytopenia.
result

  LLL accelerated platelet formation and increased platelet production of MK.

To examine the possible effects of LLL on platelet biogenesis, mature MKs were selected from mouse BM cells based on CD41 + and high forward scatter (FSChigh) and they were 810 nm diodes over different time periods (FIG. 1A). The laser was exposed. Next, the selected MK was cultured in a serum-free expansion medium (hereinafter referred to as MK medium) containing 100 ng / ml thrombopoietin (TPO) for 1 hour, and ATP measurement was performed (FIG. 1B). An LLL with an energy density range of 1-10 J / cm ² significantly enhances ATP synthesis in MK, with a marked effect of 3-5 J / cm ² (FIG. 1B). Therefore, unless otherwise specified, a 3 J / cm ² laser is selected for use in subsequent ex vivo studies. First, MKs selected with LLL or simulated light were treated, cultured in MK medium for 24 hours, and flow cytometric analysis of 5000 MK sizes was performed by FSC based on CD41 + cells. This analysis showed an average 60% increase (p <0.001, FIG. 1C) with LLL-treated MK compared to only a 37% increase in size with mock-treated MK. Next, transmission electron microscopy confirmed LLL-mediated MK increase (FIG. 1D). After 24 hours of culture, the large diameter of MK increased by 76% in the presence of LLL and increased by only 39% in the absence of LLL under similar conditions (p <0.001, FIG. 1D, lower right) ).

With the exception of cell expansion, LLL-treated MK had already produced IMS throughout the cytoplasm after 24 hours of culture (FIG. 1D, lower middle), but such membrane systems were less formed in the control MK. Showed that LLL accelerates MK maturation. IMS is a proplatelet membrane reservoir and is one of the important determinants of the number of platelets produced from each MK37. According to this, the number of platelets generated from LLL-treated MK is twice that of control MK (p <0.001, FIG. 1G), which is the MK that forms large proplatelets (PPF-MK) This is because the ratio of (1F) increased. When PPF-MK was cultured with a phase-contrast microscope for 24 hours and followed, MK converted their cytoplasm into many proplatelets decorated with multiple protrusions, which were different in size. Formed like a “flower” (FIG. 1E). In the absence of LLL, about 23.5% of CD41 + FSC ^high MK forms a large PPF-MK with a cell diameter ≧ 100 μm. Of note, when LLL is present, the percentage of large PPF-MK increases to 42.7%, which indicates an increase of more than 80% compared to sham treatment (FIG. 1F). To summarize this finding in vivo, mature MKs were selected, treated with LLL or simulated light, labeled with the biofluorescent dye carboxyfluorescein succinimidyl ester (CFSE), and recipient mouse 38 Intravenous injection. From day 2 to day 5 after injection into the receptor, LLL-treated MK produced higher levels of platelets (p <0.001, FIG. 1H) than the control counterpart.

  Important role of mitochondrial ATP production in platelet formation

Our further study showed that the coefficient of determination R ² = 0.9441 (P <0.001) and a high MK ATP level measured 1 hour after LLL between the platelet count measured 3 days later. Statistical correlation was revealed (Figure 2A). Inclusion of 5 μg / ml oligomycin A (Oligomycin A (OA)) in MK medium significantly attenuated LLL-mediated platelet production (FIG. 2B). Oligomycin A specifically suppressed mitochondrial F ₁ F ₀ -ATP synthase and decreased intracellular ATP synthesis (FIG. 2C). The importance of ATP in platelet formation is also consistent with the development of irreversible thrombocytopenia during stress in mice lacking IEX- ¹³⁹ . Compared to the wild type (WT) control, IEX-1 knockout (KO) MK has reduced mitochondrial membrane potential (Δψm, FIG. 2D) and reduced ATP production (p <0.01, FIG. 2E). Proplatelet differentiation from KO MK was markedly hindered and formed a small, short proplatelet branch with a low complexity network (FIG. 2F, middle panel). Compared to WT MK in 24 hour differentiation cultures, the average size of KO MK was reduced by half (p <0.001, FIG. 2G), demonstrating a central role of mitochondrial activity in proplatelet formation. Treatment of KOMK with LLL increased ATP levels by 89% (p <0.001, FIG. 2E). Notably, a single dose of LLL treatment substantially restored KO MK proplatelet formation, resulting in a nearly normal PPF-MK form 24 hours after LLL (FIG. 2F, right panel). ). The average diameter of KO PPF-MK was only 67.0 ± 17.8 μm but increased to 97.6 ± 31.3 μm after LLL, indicating a 46% increase in cells (P <0.01). ). However, they are still smaller than WT PPF-MK (FIG. 2G). Compared to sham treatment in 3-day cultures, LLL-mediated increase in KO PPF-MK converted to a 2-fold increase in the number of platelets produced (FIG. 2H). These data support that mitochondrial activity is a determinant of platelet production.

  LLL promotes mitochondrial biogenesis in MK.

  We next examined how a short (3 min 20 sec) LLL treatment of MK affects platelet differentiation after days. First, ATP production was measured and it was observed that LLL improved ATP production in MK only slightly, reaching a peak at 60 minutes and returning to a basal level within 90 minutes (FIG. 3A). Such instantaneous and stable ATP generation was also demonstrated in BM nucleated cells (BM), hematopoietic stem cells and progenitor cells (Lin-Sca1 + cKit + cells or LSK) after LLL treatment (Fig. 3A). Surprisingly, however, LLL-promoted mitochondrial biogenesis occurs in MK, as shown by doubling of mitochondrial content in MK alone at 24 hours after LLL compared to control. However, it did not occur in LSK or BM (FIG. 3B). Mitochondrial mass was quantified by MitoTracker staining (FIG. 3B) and relative ratio of mitochondrial DNA to nuclear DNA (FIG. 3C). LLL-mediated mitochondrial biogenesis was further demonstrated at the molecular level. In this regard, peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) is a major regulator of mitochondrial biogenesis and respiratory function. Greene et al., Physiol Rep. 3 (7). pii: e12470 (2015). The expression of this gene was strongly enhanced 4 hours after LLL (FIG. 3D), after which other genes associated with mitochondrial biogenesis were also significantly upregulated in the cell (FIG. 3D). These genes include mitochondrial transcription factor A (Tfam), mitochondrial fission related gene dynamin related protein (Drp1), mitochondrial fission 1 protein (Fis1) and mitochondrial fission factor (Mff). PGC-1α expression was significantly lower in BM cells and LSK and increased with LLL treatment. This demonstrated the ability of LLL to stimulate PGC-1α expression in different cell types, as described above. Nguyen et al., Mitochondolion. 14, 42-48 (2014). However, in contrast to MK, the Tfam, Drp1, Fis1, and Mff genes downstream of PGC-1α43 were not upregulated in BM and LSK measured in parallel. Similar to that described by Nguyen et al., This may be due to the fact that the cell diploid is different from the MK polyploid.

To confirm the important role of MK polyploidy in LLL-mediated mitochondrial biogenesis, we screened CD41 ⁺ MK from BM cells based on DNA content after staining with the biofluorescent dye Hoechst 33342 (FIG. 3E). Baccini et al., Blood 98, 3274-3282 (2001). As shown in FIGS. 3F and 3G, MKs with more than 8N DNA are much more responsive to LLL than 2N / 4N MKs, and under similar conditions, polyploid MKs over 2N / 4N MKs. Showed a marked increase in mitochondrial biogenesis and PGC-1α expression (FIG. 3F). Similar to diploid cells, the components of 2N / 4N cells contain 84% 2N MK and 16% 4N MK (mean = 2.3N), and the components of cells above 8N contain 67% Of 8N MK, 28% 16N MK and 5% 32N MK (mean = 11.4 N), considered to be polyploid MK. The DNA copy number dependent effect of LLL is more pronounced in the expression of downstream genes Tfam, Drp1, Fis1 and Mff, which were increased by 100-200% in polyploid MK but in diploid MK There was only a 0-20% increase (Figure 3H). According to this, compared to simulated light, LLL increased platelet production in polyploid MK by 200% and only 29% in diploid MK (FIG. 3I). These results indicated that LLL effectively enhanced mitochondrial biogenesis in polyploid MK, but did not enhance mitochondrial biogenesis in diploid cells.

  As shown in FIGS. 3J and 3K, an increase in mitochondrial mass was observed in multinucleated MK, where mitochondria were counted only in multinucleated cells before IMS fully developed. Interestingly, besides increasing the number of mitochondria, LLL treatment also changed the mitochondrial distribution in the cells. Mitochondria were more uniformly distributed throughout the cell (FIG. 3J, right), while mitochondria in mock-treated MKs were mainly concentrated in the perinuclear region (FIG. 3J, left). When the distance from individual mitochondria to the nearest cell nucleus was measured, 16% of mitochondria in LLL-treated MK were located at a site exceeding 4 μm from this cell nucleus, but mitochondria distant from these cell nuclei in control MK Only 4% (P <0.05, FIG. 3L). LTP-stimulated ATP production promotes faster mitochondrial migration, and to meet the energy demands of specific cell regions during MK augmentation, increasing the distance between any two mitochondria It can emit mitochondrial requirement signals that stimulate division. By inheriting, mitochondrial biogenesis can ensure increased MK and sufficient energy supply for each platelet, so as to obtain several mitochondria.

  LLL rapidly cures thrombocytopenia induced by γ-irradiation.

To explore the therapeutic potential of LLL, we first determined the laser dose that could penetrate the mouse skin, muscle and bone layers well and reach BM at 3 J / cm ² . Some lasers tested include 660 nm continuous wave lasers, 810 nm 10 Hz, 100 Hz pulsed lasers and 810 nm continuous wave lasers, the latter showing the most effective transmission, with a laser power transmitted through the BM of 9.0. ± 0.6% (FIG. 4A). Therefore, whole body irradiation is performed for 5 minutes with a 810 nm continuous wave laser with a fluence of 100 mW / cm ² or 30 J / cm ^{2 so} that mouse BM cells obtain an energy density of about 3 J / cm ² , and ex vivo culture Comparable to the laser energy used in (Fig. 1). Laser penetration was demonstrated by increased ATP production in BM cells isolated from vertebrae, femur, tibia, and pelvis 1 hour after whole-body LLL irradiation (FIG. 4B). Subsequently, confocal microscopy confirmed the effect of LLL on in vivo MK differentiation in the femur, where blood vessels and MK were stained with PE-anti-CD105 and FITC-anti-CD41 antibodies, respectively (Fig. 4C). A large “flower” -like MK, probably PPF-MK (FIG. 4C, right), was observed throughout the BM 24 hours after the LLL, but such “flower” -like cells in the BM of control mice. Was hardly seen (FIG. 4C, left figure). Quantitatively, about 36% MK formed PPF-MK in the LLL-treated femur while only 19% MK formed PPF-MK in the control femur (p <0.01, FIG. 4D).

  The above study has shown that LLL is primarily targeted at MK and has a greater impact on subjects with a large number of MKs (eg, subjects with thrombocytopenia), which causes compensatory megakaryocyte production. It showed that there is. Therefore, we induced thrombocytopenia by 3-Gy γ-irradiation (IR) (Ramsey et al., Haematologica 99, 282-291 (2014)), and then daily as described above in the following three schemes: Mice were treated with whole body LLL irradiation for 5 minutes, (1) treated once 6 hours after IR (IR + 1 x LLL), (2) treated twice 6 and 24 hours after IR (IR + 2 x LLL), (3) The treatment was performed 4 times continuously from the 0th day (IR + 4xLLL). Total blood counts are checked weekly and compared to gamma-irradiated mice receiving simulated light. Over the entire experimental period, the number of leukocytes, lymphocytes, monocytes, granulocytes or erythrocytes did not change significantly in the presence or absence of LLL. However, platelet recovery was much faster in mice depending on the laser dose (FIG. 5A). Platelet counts reached pre-IR or higher levels as early as 2 weeks after IR (IR + 4 × LLL) or 3 weeks (IR + 2 × LLL) compared to 5 weeks of sham-treated mice (IR). Consistent with the increased platelet count when checked 2 weeks after IR is the normalization of the bleeding time in the tail of the mouse (FIG. 5B) and the average platelet volume in the mouse.

  Consistent with the increased platelet count when checked 2 weeks after IR is the normalization of tail bleeding time (FIG. 5C) and the average platelet volume in mice (FIG. 5D). Platelets produced in 4 × LLL-treated mice were ultrastructurally indistinguishable from normal control platelets containing comparable levels of granules, mitochondria and open tubule system (FIG. 5E). In contrast, platelets isolated from g-irradiated mice showed an abnormal morphology (2-3 times larger than normal platelets) with small amounts of mitochondria and granules (FIG. 5E). The abnormal morphology of the platelets can be interpreted that these mice had only a 40% reduction in platelet count compared to normal controls, but the bleeding time doubled (FIGS. 5, B and C). The overall aggregation activity of platelets isolated from 4 × LLL-treated mice was also identical to that of normal controls that did not receive IR (FIG. 5F). These results indicated that the platelets produced by the LLL treatment remained morphologically and functionally intact.

  Although LLL enhanced platelet production in thrombocytopenic mice, it did not significantly affect platelet counts in normal mice when administered every other day for up to 12 days compared to sham-treated mice. (FIG. 5G). The number of MKs also did not change significantly (FIG. 5G). This supports the safety of such methods, and there is little concern about thrombus formation even after repeated use of LLL.

  Besides increasing and accelerating proplatelet formation, LLL can also protect MK from IR-induced apoptosis, and during the first 3 days after IR, the number of MKs in LLL-treated mice is mock-treated. Resulted in more than the MK in the treated mice. In the presence of LLL, the number of MK peaks 2 days after IR and increases from 37353 to 78159 in one femur, which is about 50% higher than in the absence of LLL (FIG. 5H). When MKs were selected, 3-Gy IR was performed, and caspase-3 / 7 activation was measured, for non-IR counterparts, g-irradiated MKs averaged caspase-3 / A 3-fold increase in 7 activity was achieved and cell viability was significantly reduced simultaneously within 24 hours after IR. LLL administered 6 hours after IR markedly suppressed caspase-3 activation and enhanced cell viability of γ-irradiated MK. LLL-mediated protection of MK from IR-induced damage increased the percentage of total PPF-MK from 20.5% to 30.2%, especially the large percentage of PPF-MK (5.8 to 19.2% And increased recovery of platelet production of γ-irradiated MK cultured ex vivo. It should be noted that the number of MK sharply dropped to the lowest level on day 5 after IR after the first increase (FIG. 5H). However, in LLL-treated mice, the number of MKs steadily increased again and the number of MKs in sham-treated mice continued to decrease (FIG. 5H), which is better than MKs from HSCs in response to LLL May be due to poor differentiation and further research is needed to reach this conclusion.

  LLL reduces thrombocytopenia induced by anti-CD41 antibodies or 5-fluorouracil.

We further expanded our work on ITP to exclude that LLL-mediated platelet production is specific for γ-irradiation-induced thrombocytopenia. From day 0 to day 7, anti-CD41 antibody was administered daily at 0.1 mg / kg body weight to deplete platelets and create a commonly used ITP animal model. Katsman et al., Transfusion 50, 1285-1294 (2010). Mice were treated daily with simulated light or 30 J / cm ² LLL, initial irradiation was performed on day 3 when the platelet count was significantly reduced, and the platelet count was checked daily 6 hours after LLL (FIG. 6A). Ultimately, all mouse platelet counts recovered due to compensatory platelet production, but after only 2 treatments (day 4) LLL lifted the lowest point and platelet count recovery in the presence of anti-CD41 antibody Was accelerated (FIG. 6A). In animals treated with LLL, bleeding time was also normalized on day 5 (FIG. 6B). Since ITP is a common form of thrombocytopenia, the ability of LLL to enhance platelet regeneration in the presence of anti-CD41 antibodies has greatly expanded its application. A similar effect of LLL on platelet regeneration was also observed in mice that received 5-FU. Chemotherapeutic drugs reduced circulating platelet count by 43% at a dose of 150 mg / kg body weight on day 4 (Chenaille et al., Blood 76, 508-515 (1990)), but daily from day 0 to day 2. Administration of three doses of LLL at a time greatly reduced thrombocytopenia (FIG. 6C) and normalization bleeding time (FIG. 6D) in drug-treated mice.

  LLL shows the potential for thrombus formation in human cells.

We subsequently evaluated the translational potential of this form using human cells. CD34 + cells were cultured in serum-free expansion medium containing 100 ng / ml human TPO, which reproduced all differentiation stages of megakaryocyte formation as described above. Zeuner et al., Cancer Res. 67, 4767-4773 (2007). As shown in FIG. 7A, in culture, CD34 + cells differentiated mainly into MK progenitor cells in 6 days, differentiated into MK in 12 days, and differentiated into platelets in 15 days. Therefore, we selected mature MK from day 12 cultures and treated the cells with different energy densities of LLL. LLL with an energy density of 0.5-10 J / cm ² (peak response is 3 J / cm ² ) significantly stimulates ATP production in human MK (FIG. 7B), similar to mouse MK (FIG. 1B). Therefore, the same laser (3 J / cm ² ) was administered on day 0 (mainly CD34 + cells in culture), day 6 (MK progenitor cells) or day 12 (MK) and then on day 15 Platelet production was evaluated. MK differentiation in these cultures was demonstrated by increasing polyploids over time (maximum percentage of polyploid cells (≧ 8N) on day 12) (FIG. 7C). Increased cell ploidy was associated with the effect of LLL on platelet production, with platelet production induced by LLL treatment reaching the highest level on day 12 of culture (FIG. 7D). The results clearly showed that MK is a preferential target for LLL in mice and that LLL has a similar effect on the development of platelet organisms between human and mouse MKs.
Debate

  Current studies demonstrate that non-invasive LLL irradiation can steadily increase platelet production in thrombocytopenic mice, but not in normal controls. The laser is equally effective in ex vivo human and mouse MKs, consistent with mitochondrial evolutionary conservation and platelet biogenesis between these two species. This observation proves convincingly about the translational potential of LLL as a treatment and prevention of thrombocytopenia. The most important finding of this study is that LLL primarily targets MK, which maintains LLL-mediated platelet production under the check of free plasma TPO (inversely correlated with the number of circulating platelets). Shinjo et al., Leukemia 12, 295-300 (1998). In contrast, all current drugs used clinically or in developmental conditions to treat thrombocytopenia promote the differentiation of MK precursors from HSCs independent of platelet count, and thus when administered at high doses Poses a high risk of thrombus formation. Since the number of MKs is reciprocally regulated by platelet count via megakaryocyte production, the more severe thrombocytopenia, the stronger megakaryocyte production is induced, large amounts of MK are produced, and LLL for platelet formation The effect becomes more pronounced. Conversely, when under physiological conditions or when platelet counts return to normal levels, LLL has little effect on platelet counts due to the very low number of MKs in these healthy subjects (FIG. 5E). Theoretically, LLL is beneficial to all patients with acquired thrombocytopenia, regardless of its etiology, and the condition is that IR, ITP and 5-FU-induced thrombocytopenia have been identified in current studies. Although being tested, thrombocytopenia can severely induce megakaryocyte production. However, at present, it is not clear whether such a strategy will affect hereditary thrombocytopenia as well. For patients with inadequate megakaryocyte production, combinations of LLL and megakaryocyte drugs (eg, recombinant human interleukin-11 (rHuIL-11), romiprostim and eltrobopag) are cumulative or cooperatively Biogenesis can be enhanced and dose-dependent side effects of these drugs can be reduced (Vadhan-Raj, S., Semin. Hematol., 46, S26-S32 (2009)), which This is because it targets an early differentiation stage of platelet production different from LLL. As far as we know, there are currently no downstream drugs that specifically target proplatelet formation or meganucleation.

  The mechanism of LLL-mediated platelet production depends primarily on its unique action on mitochondria. LLL protects MK from apoptosis induced by γ-irradiation. γ-irradiation induces apoptosis through a mitochondrial-dependent pathway, as demonstrated by many studies. Sridharan et al., Radiat. Res. 181, 324-334 (2014). Second, LLL specifically enhanced mitochondrial biogenesis in MK (FIG. 3), which is not shown in other cell types and is due to MK ploidy (a unique feature of MK). . Previous studies showed that near-infrared light exposure increased PGC-1α expression in muscle cells by approximately 20%, but the expression of downstream mitochondrial component genes (Tfam, NRF-1, Sirt3 and cytochrome c) did not change It showed that. Similarly, LLL increased PGC-1α transcription in BM and LSK but not with mitochondrial biogenesis (FIG. 3B) or with the expression of other mitochondrial component genes. This unique effect of LLL on mitochondrial biogenesis in polyploid MK is consistent with a functional expansion that increases the MK genome required for protein synthesis associated with platelet function and parallels cell expansion. Raslova et al., Blood, 101, 541-544 (2003). The specific MK effect of LLL well explains that LLL greatly affects MK and has little effect on BM and LSK (FIG. 3B), lymphocytes and red blood cells under similar conditions.

  The ability of LLL to enhance MK mitochondrial biogenesis and mitochondrial activity is thought to be essential for its platelet producing effect (Mostafa et al., Exp. Hematol. 29, 873-883 (2001)). This is deduced from the correlation between ATP production and platelet production (FIG. 2A). The high energy demand for proplatelet formation is also consistent with in vivo platelet biogenesis, where MK transitions to the BM sinusoid during proplatelet formation, where oxygen levels increase and massive mitochondrial oxidative phosphorylation HSCs that are predominantly present in low-oxygen niches in bone and progenitor cells conflict. Similarly, MK tends to form proplatelets in lung capillaries that contain high levels of oxygen. In contrast, insufficient activity of mitochondria lacking IEX-1 prevents proplatelet formation, which can be markedly normalized by LLL treatment (FIG. 2). Direct evidence of the importance of ATP in proplatelet formation comes from the work of Richardson and Patel et al. Richardson et al., Blood 106, 4066-4075 (2005). They showed that adding ATP to PPF-MK permeabilized with Triton X-100 activated proplatelet elongation and significantly enhanced proplatelet growth. This study not only provides convincing evidence for the rate-limiting factor of ATP in the late stages of thrombus formation, but also provides valuable suggestions on how to improve platelet production effects both in vitro and in vivo.

LLL therapy has already been used in clinical practice for more than 20 years for analgesia, anti-inflammatory and wound healing and has a long-term safety record. Most practitioners can easily adopt such a safe, drug-free, donor-independent manner as a stand-alone or complementary treatment for thrombocytopenia. With respect to laser irradiation in the human body, there is no danger of heating and the superpulse infrared laser can penetrate the tissue up to 10-13 cm. Therefore, what is worth studying is whether Superpulse LLL can non-invasively increase platelet biogenesis in large animals in the near future. It should be emphasized that such a scheme is not intended to replace platelet injection in blood loss treatment, greatly reducing the demand for platelet injection and providing primary or secondary prevention of thrombocytopenia.
Materials and methods

  Research design

  This study aims to determine the definitive effects of LLL on platelet biogenesis and its therapeutic and preventive potential for thrombocytopenia. For all ex vivo studies we used primary MK selected from cultures derived from mouse BM or CD34 + cells. The number of experiments (including biological and technical replication) is defined in the legends of each figure. For in vivo experiments, three different mouse models were tested to verify the ability of LLL to cure or ameliorate thrombocytopenia induced by irradiation, immunodepletion and 5-FU. The number of mice is outlined in the legend for each figure. Researchers do not know the characteristics of the sample. All outliers studied are included in the data analysis.

  mouse

  C57BL / 6 mice of any gender 8-12 weeks old were purchased from Jackson Laboratory. 129Sv / C57BL / 6 background WT and IEX-1 KO mice were generated in our laboratory. Zhang et al. Cereb. Blood Flow Metab 34, 1391-1401 (2014). In accordance with the National Institutes of Health guidelines on the care and use of laboratory animals, animal protocols were approved by the Research Animal Care of the Massachusetts General Hospital at the Massachusetts General Hospital. .

  Low level laser treatment

For ex vivo irradiation, in order to obtain an energy density of ³ J / cm ² , an 810 nm infrared diode laser (Accelerator, PhotoThera) was used as a continuous wave, and the output density was 15 mW / cm ² and continued for 3 minutes and 20 seconds. For whole-body LLL irradiation, mice depilated with isoflurane were anesthetized and placed under a laser lens covering the trunk and the entire limb. In order to obtain an energy density of 30 J / cm ² , the LLL power density is 100 mW / cm ² and the total exposure time is 5 minutes. The first dose of LLL was given 4-6 hours after IR or 5-FU treatment, or 3 days after the first injection of anti-CD41 antibody. Simulated light was applied using a small soft white LED bulb (3W, A15) from General Electric. Whenever laser power transmission was measured, fresh skin, muscle and vertebral layers were removed immediately after the mice were killed, immediately after exposure to different lasers. Transmitted light was measured with a laser power meter (Ophir Nova II), and the difference in light energy density between the skin surface and the bone layer was calculated as transmittance (%).

  Proplatelet formation assay

CD41 + FSC _high MK was selected, treated with or without LLL, and placed in MK medium supplemented with 3.75 g / L methylcellulose (Sigma). Cells were differentiated in a chamber containing 5% CO ₂ at 37 ° C. Phase contrast live cell images were recorded for up to 24 hours using a 40 × objective with a time-lapse microscope (Zeiss Axio Observer Z1). The longest or large diameter of PPF-MK was measured by AxioVision software (Zeiss). PPF-MK with a diameter <100 μm is defined as “small”, and one with a diameter ≧ 100 μm is defined as “large”. To estimate the rate of PPF-MK formation, 500 CD41 + FSC _high MKs were placed in each hole, and the percentage of PPF-MK was manually determined from at least 6 samples per group by an unknowing researcher. Calculated.

  Tracking of femur MK

  Twenty-four hours after whole body LLL irradiation, 12 μg of FITC anti-CD41 and 12 μg of PE anti-CD105 antibody (BioLegend) were administered intravenously per mouse. After 15 minutes, the mice were killed and the femurs were removed and examined with a confocal microscope. At least 50 MKs were tracked in 6 randomly selected views from each femur, and the percentage of PPF-MK was calculated from 6 samples per group in a sample-blind manner.

  Human megakaryocyte and platelet cultures

  Frozen human BM CD34 + cells were obtained from STEMCELL Technologies and differentiated in serum-free expansion medium supplemented with 100 ng / ml human TPO (STEMCELL Technologies) as described above. Zeuner et al., Cancer Res. 67, 4767-4773 (2007). During the culture, the differentiation stage of megakaryocytes was routinely assessed by May-Grunwald-Giemsa staining (Sigma) and by flow cytometry CD41 levels. CD34 + cells, MK progenitor cells, mature MK or platelets were collected on day 0, 6, 12, or 15 of culture, respectively.

  Statistical analysis

Results are shown as mean ± SEM. Statistical saliency was assessed by two-tailed Student's t-test (used for comparison between two groups) or by one-way analysis of variance (used for comparing multiple groups). A value of p <0.05 is considered statistically significant. The relationship between ATP levels and platelets was measured by regression analysis and correlation analysis, and the coefficient of determination (R ² ) was calculated. All statistical analyzes were performed using GraphPad Prism 6.0 (GraphPad Software).
Example 2: Drug that promotes mitochondrial biogenesis for platelet regeneration

  Mitochondrial biogenesis has been extensively studied for decades, mainly in tissues with high energy demand, such as heart, liver, skeletal muscle, fat, and brain. Megakaryocytes (MK) are also rich in mitochondria at the final differentiation stage, but the importance of mitochondrial biogenesis in platelet biogenesis was discovered until our recent work. Yang et al., Sci. Rep. 6: 38238 (2016), Zhang et al., Sci. Transl. Med. 8: 349ra101 (2016). We demonstrate that at the final stage of platelet formation, massive cytoplasmic remodeling and active overhang and elongation of proplatelets are highly dependent on mitochondrial activity. As a support, mitochondrial cytochrome c point mutations cause dysregulation of platelet formation and thrombocytopenia in humans, and other diseases do not occur simultaneously in the family. Morison et al., Nat. Genet. 40: 387-389 (2008). We have also demonstrated that mitochondrial dysfunction is prone to thrombocytopenia during stress in mice. Ramsey et al., Haematologica 99: 282-291 (2014). Conversely, low level laser therapy (LLLT) promotes mitochondrial biogenesis and platelet formation in MK and reduces thrombocytopenia in multiple mouse models. These findings raise the interesting possibility that mitochondrial biogenesis promoting drugs (collectively referred to herein as mitochondrial drugs (mitochondrrugs)) may be able to enhance platelet regeneration and treat thrombocytopenia. To do.

  As shown in FIG. 8, mitochondrial biogenesis can be treated pharmacologically by inducing the expression of peroxisome proliferator-activated receptor (PPAR) -γ coactivator 1α (PGC-1α). The peroxisome proliferator-activated receptor (PPAR) -γ coactivator 1α (PGC-1α) is a master regulatory gene for mitochondrial biogenesis. Scarpulla, R.M. C. Biochim. Biophys. Acta 1813: 1269-1278 (2011). This gene is transcriptionally activated by various kinases and PPAR agonists, or phosphorylated using AMP-activated kinase (AMPK) and deacetylated using silent information regulator 2 protein 1 (SIRT1) (De-Ac) Can be post-translationally modified (FIG. 8). Komen et al., Br. J. et al. Pharmacol. 171: 1818-1836 (2014). Numerous studies have shown that mitochondrial biosynthesis activates AMPK such as bezafibrate (BEZ), rosiglitazone, pioglitazone and fenofibrate, including pan-PPAR agonists such as AICAR (AMP mimic), metformin, and possibly resveratrol. It has been shown that it can be sufficiently induced by activators such as agents such as SRT1720, its derivatives SRT2183 and SRT1460, quercetin, and possibly resveratrol SIRT1. Uittenbogaard, M.M. and Chiaramello, A.A. Curr. Pharm. Des, 20: 5574-5593 (2014). Arbel et al., Cardiovasc. Diabetol. 15:11 (2016). Some of these mitochondrial drugs are currently used in clinical experiments to treat metabolic syndrome, obesity, Duchenne muscular dystrophy and various neurodegenerative diseases, but other drugs (e.g., BEZ, metformin) has already been used clinically for decades. Hofer et al., Hum. Mol. Genet. 23: 2400-2415 (2014). However, the ability of these drugs alone or in any combination to enhance mitochondrial biogenesis or platelet production in MK has not been studied.

  Some mitochondrial drugs tested enhanced ex vivo MK platelet production. We have found that resveratrol (Res), BEZ and SRT1720, although slightly less than LLLT, can significantly enhance ex vivo MK platelet production and not AICAR (FIG. 9). The concentrations used for individual drugs are those commonly used to induce PGC-1α expression in other cell cultures, with the exception of SRT1720, which is used at 100-fold lower concentrations. High concentrations of SRT1720 cannot significantly enhance platelet production. Various drug concentrations may be optimized to enhance platelet production. These mitochondrial drugs have also been shown to be advantageous for chemotherapy (Aires et al., Mol. Nutr. Food Res. 58: 1785-1794 (2014), Liu et al., J. Cancer 6: 1214-1221, (2015). , Fresco et al., Curr. Pharm. Des 16: 114-134 (2010)), further research is needed to conclude, but may provide dual benefit to cancer patients undergoing chemotherapy it can.

  Although there is a certain delay, mitochondrial drugs enhance platelet production in vivo similar to LLLT. To demonstrate mitochondrial drug-mediated platelet biogenesis in vivo, 8-week-old B6 mice received 2 doses of chemical drug 5 -Fluorouracil (5-FU) was administered. To induce thrombocytopenia, 120 and 90 mg / kg body weight were administered on days 1 and 4, respectively (FIG. 9). From 6 hours after the first 5-FU injection, mice were treated with 100 mg / kg body weight BEZ or Res or solvent control by oral gavage to mice treated with 5-FU twice a day for 4 consecutive days. For comparison, LLL was administered daily for 4 consecutive days in parallel to 5-FU-treated mice. BEZ showed the effectiveness of retaining a similar plt number as LLLT after 7 and 7 days, but was inferior to LLLT before 7 days, probably because LLLT removed MK and platelets from apoptosis. This is because it was protected and BEZ did not. Despite being delayed compared to LLLT, BEZ maintained platelet counts above the non-risk level of platelet count (70% of normal) and substantially reduced the lowest point. This is the point where the lowest score results in the most dangerous bleeding. Res also enhanced platelet biogenesis and markedly raised the lowest point, but its effectiveness was relatively weak compared to BEZ or LLLT. The results demonstrate that induction of mitochondrial biogenesis can reduce thrombocytopenia. The dose of Res or BEZ in the study is comparable to the current dose of mitochondrial drugs in the clinic. Whether higher doses or doses of BEZ can further improve efficacy has not yet been studied.

  The combination of LLLT and mitochondrial drugs can expand the benefits. The rapid action of LLLT and the convenience of oral BEZ facilitate the combination of the two to treat anti-CD41 antibody-induced thrombocytopenia (a commonly used immune thrombocytopenia (ITP) model) It was done. Antiplatelet antibodies were administered daily from day 0 to day 7, which caused the circulating platelets to drop sharply after the second injection, causing the lowest point on day 2. During the 8 days of the entire experiment, platelet levels remained below 40% of the normal platelet count. Single BEZ could not effectively prevent the platelet count from dropping to the lowest point, but it significantly increased the platelet count immediately after the lowest point. In striking contrast, LLLT maintained platelet count levels higher than 50% of normal levels, greatly reducing the risk of bleeding. The combination of BEZ and LLLT further improved platelet regeneration, maintaining the platelet count above 70% of the normal level (safe level of circulating platelet count). These results indicate the potential of mitochondrial drugs to treat thrombocytopenia when mitochondrial drugs are used alone or in combination with LLLT or other facilitating drugs that promote megakaryocyte production. These results are shown in FIGS.

  The complete disclosure of all patents, patent applications, and publications, and electronically available materials cited herein are hereby incorporated by reference. Any inconsistencies between the material incorporated herein by reference and the present specification will be resolved in a manner advantageous to this specification. The foregoing detailed description and examples have been given for clarity of understanding only. Unnecessary restrictions should then not be understood. The invention is not limited to the specific details shown and described, as variations obvious to one skilled in the art are included within the scope of the invention as defined by the claims.

Claims (11)

  A method of stimulating platelet formation in a body of a subject by administering to the subject an effective amount of a drug that stimulates mitochondrial biogenesis.   The drugs include p38 mitogen activated protein kinase activator, calmodulin-dependent protein kinase IV activator, AMP activated kinase activator, calcineurin A activator, peroxisome proliferator response element activator and SIRT1 activation 2. A method according to claim 1 selected from the group consisting of agents.   2. The drug that stimulates mitochondrial biogenesis is selected from the group consisting of bezafibrate, rosiglitazone, pioglitazone, fenofibrate, AICAR, metformin, resveratrol, SRT1720, SRT2183, SRT1460 and quercetin, and derivatives thereof. The method described in 1.   The method of claim 1, wherein the subject has been diagnosed as having thrombocytopenia.   5. The method of claim 4, further comprising administering an antithrombocytopenic agent to the subject.   The method of claim 1, further comprising treating the subject with low level light (LLL) therapy.   The method of claim 1, wherein the drug is administered with a pharmaceutically acceptable carrier. A method of stimulating the formation of platelets,
Contacting the platelet precursor with a drug that stimulates mitochondrial biogenesis.   The method according to claim 8, wherein the platelet precursor is a megakaryocyte or a megakaryoblast.   9. The method of claim 8, wherein the platelet precursor is also exposed to low level light therapy.   9. The method of claim 8, wherein the platelet precursor is in vitro or ex vivo.

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